NR was extracted by using 4 volumes of ice-cold 50 mM pH 7.8 MOPS-KOH buffer containing 5 mM EDTA, 5 mM NaF, 2 mM MSH, 1 mM PMSF, 10 μM FAD, 1 μM leupeptin and 10 μM chymostatin.
Cells (∼2 × 10) in 2 mL of ice-cold disruption buffer (PBS containing 0.1 mM EDTA, 10% glycerol, 0.2 mM FAD, 1 μg/mL protease inhibitor cocktail, and 0.1 mM PMSF) were disrupted by sonication (2 × 10 s) in a bath sonicator at 3 °C, according to the previously described methods (30).
Lyophilized whole E. coli BL21 DE3) cell preparations containing overexpressed genes of ω-TA_Cv 18 (20 mg) and LCAO_Af (40 mg) were both resuspended in sodium phosphate buffer (500 μL each, 100 m m, pH 10) supplemented with pyridoxal-5′-phosphate (PLP, 2 m m), NAD+ (2 m m) and FAD (1 m m).
In order to obtain a structure with bound FAD, it therefore proved necessary to soak the HbpA crystals in a solution of the mother liquor with added FAD (1 m m), as well as 20%% ethylene glycol (as cryoprotectant) for 5 min prior to flash-cooling in liquid nitrogen for X-ray diffraction analysis.
Adenovirus infection (2.6 × 10 p.f.u./insert in a 12-well plate) was carried out on day 1 after seeding onto collagen type I gels in a mixture of conditioned MCDB153 supplemented with 10% dFCS and FAD (1 : 1 volume) for 24 h.
From 25 to 30 mg of total lysate protein, the combined NEF and LSP were resuspended by homogenization on ice in buffer B [25 mM Hepes (pH 7.4) with 130 mM NaCl, 0.1 mM MgCl2, 10% glycerol, 1 μg/mL protease inhibitor cocktail, 0.5 mM PMSF, 20 μM FAD, 1% Nonidet P-40, and 0.2% deoxycholate] and then stirred gently for 30 min at 4 °C.
Sakha 53 at wider spacing of 25 cm with application of nitrogen fertilization levels of 45 N fad.−1.−1
The wider plant spacing of 25 cm seems to be a good compromise between the highest seed yield fad.−1 and good acid composition of oil.
The highest values of head diameter, 100-seed weight, seed weight plant−1 and hence seed yield fad.−1 as well as the highest proportion of oleic and linoleic unsaturated fatty acids composition were obtained by grown sunflower cv.
Sakha 53 was ranked in the first order in stem diameter, head diameter, 100-seed weight, seed weight plant−1, flowered late and hence seed yield fad.−1 as well as seed oil content whereas, Giza 102 characterized with its contained the highest proportion of oleic and linoleic unsaturated fatty acids.
In both seasons, the experiment was conducted using the split split plot design in randomized complete block design with three replicates arrangement keeping plant spaces (15, 20 and 25 cm apart between hills) in main plots, nitrogen fertilization levels (15, 30 and 45 N fad.−1) in sub plots and sunflower genotypes (Giza 102, Sakha 53 and promising line of L120) in sub sub plots.
